Heterogeneity of ferrous iron-containing endolysosomes and effects of endolysosome iron on endolysosome numbers, sizes, and localization patterns.

Endolysosomes are key regulators of iron metabolism and are central to iron trafficking and redox signaling. Iron homeostasis is linked to endolysosome acidity and inhibition of endolysosome acidity triggers iron dysregulation. Because of the physiological importance and pathological relevance of ferrous iron (Fe2+ ), we determined levels of Fe2+ specifically and quantitatively in endolysosomes as well as the effects of Fe2+ on endolysosome morphology, distribution patterns, and function. The fluorescence dye FeRhoNox-1 was specific for Fe2+ and localized to endolysosomes in U87MG astrocytoma cells and primary rat cortical neurons; in U87MG cells the endolysosome concentration of Fe2+ ([Fe2+ ]el ) was 50.4 µM in control cells, 73.6 µM in ferric ammonium citrate (FAC) treated cells, and 12.4 µM in cells treated with the iron chelator deferoxamine (DFO). Under control conditions, in primary rat cortical neurons, [Fe2+ ]el was 32.7 µM. Endolysosomes containing the highest levels of Fe2+ were located perinuclearly. Treatment of cells with FAC resulted in endolysosomes that were less acidic, increased in numbers and sizes, and located further from the nucleus; opposite effects were observed for treatments with DFO. Thus, FeRhoNox-1 is a useful probe for the study of endolysosome Fe2+ , and much more work is needed to understand better the physiological significance and pathological relevance of endolysosomes classified according to their heterogeneous iron content Cover Image for this issue: https://doi.org/10.1111/jnc.15396.

Mechanisms of neuronal dysfunction in HIV-associated neurocognitive disorders.

HIV-associated neurocognitive disorder (HAND) is characterized by cognitive and behavioral deficits in people living with HIV. HAND is still common in patients that take antiretroviral therapies, although they tend to present with less severe symptoms. The continued prevalence of HAND in treated patients is a major therapeutic challenge, as even minor cognitive impairment decreases patient's quality of life. Therefore, modern HAND research aims to broaden our understanding of the mechanisms that drive cognitive impairment in people with HIV and identify promising molecular pathways and targets that could be exploited therapeutically. Recent studies suggest that HAND in treated patients is at least partially induced by subtle synaptodendritic damage and disruption of neuronal networks in brain areas that mediate learning, memory, and executive functions. Although the causes of subtle neuronal dysfunction are varied, reversing synaptodendritic damage in animal models restores cognitive function and thus highlights a promising therapeutic approach. In this review, we examine evidence of synaptodendritic damage and disrupted neuronal connectivity in HAND from clinical neuroimaging and neuropathology studies and discuss studies in HAND models that define structural and functional impairment of neurotransmission. Then, we report molecular pathways, mechanisms, and comorbidities involved in this neuronal dysfunction, discuss new approaches to reverse neuronal damage, and highlight current gaps in knowledge. Continued research on the manifestation and mechanisms of synaptic injury and network dysfunction in HAND patients and experimental models will be critical if we are to develop safe and effective therapies that reverse subtle neuropathology and cognitive impairment.

Orai1 Plays a Crucial Role in Central Sensitization by Modulating Neuronal Excitability.

Pathological pain is a common and debilitating condition that is often poorly managed. Central sensitization is an important mechanism underlying pathological pain. However, candidate molecules involved in central sensitization remain unclear. Store-operated calcium channels (SOCs) mediate important calcium signals in nonexcitable and excitable cells. SOCs have been implicated in a wide variety of human pathophysiological conditions, including immunodeficiency, occlusive vascular diseases, and cancer. However, the role of SOCs in CNS disorders has been relatively unexplored. Orai1, a key component of SOCs, is expressed in the human and rodent spinal cord dorsal horn, but its functional significance in dorsal horn neurons is poorly understood. Here we sought to explore a potential role of Orai1 in the modulation of neuronal excitability and A-type potassium channels involved in pain plasticity. Using both male and female Orai1 knock-out mice, we found that activation of Orai1 increased neuronal excitability and reduced A-type potassium channels via the protein kinase C-extracellular signal-regulated protein kinase (PKC-ERK) pathway in dorsal horn neurons. Orai1 deficiency significantly decreased acute pain induced by noxious stimuli, nearly eliminated the second phase of formalin-induced nociceptive response, markedly attenuated carrageenan-induced ipsilateral pain hypersensitivity and abolished carrageenan-induced contralateral mechanical allodynia. Consistently, carrageenan-induced increase in neuronal excitability was abolished in the dorsal horn from Orai1 mutant mice. These findings uncover a novel signaling pathway involved in the pain process and central sensitization. Our study also reveals a novel link among Orai1, ERK, A-type potassium channels, and neuronal excitability.SIGNIFICANCE STATEMENT Orai1 is a key component of store-operated calcium channels (SOCs) in many cell types. It has been implicated in such pathological conditions as immunodeficiency, autoimmunity, and cancer. However, the role of Orai1 in CNS disorders remains poorly understood. The functional significance of Orai1 in neurons is elusive. Here we demonstrate that activation of Orai1 modulates neuronal excitability and Kv4-containing A-type potassium channels via the protein kinase C-extracellular signal-regulated protein kinase (PKC-ERK) pathway. Genetic knock-out of Orai1 nearly eliminates the second phase of formalin-induced pain and markedly attenuates carrageenan-induced pain hypersensitivity and neuronal excitability. These findings reveal a novel link between Orai1 and neuronal excitability and advance our understanding of central sensitization.

Induction of Interleukin-1ß by Human Immunodeficiency Virus-1 Viral Proteins Leads to Increased Levels of Neuronal Ferritin Heavy Chain, Synaptic Injury, and Deficits in Flexible Attention.

Synaptodendritic pruning and alterations in neurotransmission are the main underlying causes of HIV-associated neurocognitive disorders (HAND). Our studies in humans and nonhuman primates indicated that the protein ferritin heavy chain (FHC) is a critical player in neuronal changes and ensuing cognitive deficit observed in these patients. Here we focus on the effect of HIV proteins and inflammatory cytokines implicated in HAND on neuronal FHC levels, dendritic changes, and neurocognitive behavior. In two well characterized models of HAND (HIV transgenic and gp120-treated rats), we report reductions in spine density and dendritic branches in prefrontal cortex pyramidal neurons compared with age-matched controls. FHC brain levels are elevated in these animals, which also show deficits in reversal learning. Moreover, IL-1ß, TNF-α, and HIV gp120 upregulate FHC in rat cortical neurons. However, although the inflammatory cytokines directly altered neuronal FHC, gp120 only caused significant FHC upregulation in neuronal/glial cocultures, suggesting that glia are necessary for sustained elevation of neuronal FHC by the viral protein. Although the envelope protein induced secretion of IL-1ß and TNF-α in cocultures, TNF-α blockade did not affect gp120-mediated induction of FHC. Conversely, studies with an IL-1ß neutralizing antibody or specific IL-1 receptor antagonist revealed the primary involvement of IL-1ß in gp120-induced FHC changes. Furthermore, silencing of neuronal FHC abrogates the effect of gp120 on spines, and spine density correlates negatively with FHC levels or cognitive deficit. These results demonstrate that viral and host components of HIV infection increase brain expression of FHC, leading to cellular and functional changes, and point to IL-1ß-targeted strategies for prevention of these alterations. Significance statement: This work demonstrates the key role of the cytokine IL-1ß in the regulation of a novel intracellular mediator [i.e., the protein ferritin heavy chain (FHC)] of HIV-induced dendritic damage and the resulting neurocognitive impairment. This is also the first study that systematically investigates dendritic damage in layer II/III prefrontal cortex neurons of two different non-infectious models of HIV-associated neurocognitive disorders (HAND) and reveals a precise correlation of these structural changes with specific biochemical and functional alterations also reported in HIV patients. Overall, these data suggest that targeting the IL-1ß-dependent FHC increase may represent a valid strategy for neuroprotective adjuvant therapies in HAND.

STIMs and Orai1 regulate cytokine production in spinal astrocytes.

BACKGROUND: Our previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes. METHODS: Primary cultured astrocytes were prepared from neonatal (P2-P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay. RESULTS: We found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability. CONCLUSIONS: This study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.

Native store-operated calcium channels are functionally expressed in mouse spinal cord dorsal horn neurons and regulate resting calcium homeostasis.

Store-operated calcium channels (SOCs) are calcium-selective cation channels that mediate calcium entry in many different cell types. Store-operated calcium entry (SOCE) is involved in various cellular functions. Increasing evidence suggests that impairment of SOCE is responsible for numerous disorders. A previous study demonstrated that YM-58483, a potent SOC inhibitor, strongly attenuates chronic pain by systemic or intrathecal injection and completely blocks the second phase of formalin-induced spontaneous nocifensive behaviour, suggesting a potential role of SOCs in central sensitization. However, the expression of SOCs, their molecular identity and function in spinal cord dorsal horn neurons remain elusive. Here, we demonstrate that SOCs are expressed in dorsal horn neurons. Depletion of calcium stores from the endoplasmic reticulum (ER) induced large sustained calcium entry, which was blocked by SOC inhibitors, but not by voltage-gated calcium channel blockers. Depletion of ER calcium stores activated inward calcium-selective currents, which was reduced by replacing Ca(2+) with Ba(2+) and reversed by SOC inhibitors. Using the small inhibitory RNA knockdown approach, we identified both STIM1 and STIM2 as important mediators of SOCE and SOC current, and Orai1 as a key component of the Ca(2+) release-activated Ca(2+) channels in dorsal horn neurons. Knockdown of STIM1, STIM2 or Orai1 decreased resting Ca(2+) levels. We also found that activation of neurokinin 1 receptors led to SOCE and activation of SOCs produced an excitatory action in dorsal horn neurons. Our findings reveal that a novel SOC signal is present in dorsal horn neurons and may play an important role in pain transmission.

The US9-Derived Protein gPTB9TM Modulates APP Processing Without Targeting Secretase Activities.

Alteration of neuronal protein processing is often associated with neurological disorders and is highly dependent on cellular protein trafficking. A prime example is the amyloidogenic processing of amyloid precursor protein (APP) in intracellular vesicles, which plays a key role in age-related cognitive impairment. Most approaches to correct this altered processing aim to limit enzymatic activities that lead to toxic products, such as protein cleavage by ß-secretase and the resulting amyloid ß production. A viable alternative is to direct APP to cellular compartments where non-amyloidogenic mechanisms are favored. To this end, we exploited the molecular properties of the herpes simplex virus 1 (HSV-1) transport protein US9 to guide APP interaction with preferred endogenous targets. Specifically, we generated a US9 chimeric construct that facilitates APP processing through the non-amyloidogenic pathway and tested it in primary cortical neurons. In addition to reducing amyloid ß production, our approach controls other APP-dependent biochemical steps that lead to neuronal deficits, including phosphorylation of APP and tau proteins. Notably, it also promotes the release of neuroprotective soluble αAPP. In contrast to other neuroprotective strategies, these US9-driven effects rely on the activity of endogenous neuronal proteins, which lends itself well to the study of fundamental mechanisms of APP processing/trafficking. Overall, this work introduces a new method to limit APP misprocessing and its cellular consequences without directly targeting secretase activity, offering a novel tool to reduce cognitive decline in pathologies such as Alzheimer's disease and HIV-associated neurocognitive disorders.

The Endolysosomal Transporter DMT1 is Required for Morphine Regulation of Neuronal Ferritin Heavy Chain.

NeuroHIV and other neurologic disorders present with altered iron metabolism in central nervous system neurons. Many people with HIV also use opioids, which can worsen neuroHIV symptoms by further dysregulating neuronal iron metabolism. Our previous work demonstrated that the µ-opioid agonist morphine causes neuronal endolysosomes to release their iron stores, and neurons respond by upregulating ferritin heavy chain (FHC), an iron storage protein associated with cognitive impairment in neuroHIV. Here, we investigated if this process required divalent metal transporter 1 (DMT1), a well-known iron transporter expressed on endolysosomes. We first optimized conditions to detect DMT1 isoforms (DMT1 1B ± iron responsive element) using fluorescently labeled rat DMT1 constructs expressed in HEK-293 cells. We also expressed these constructs in primary rat cortical neurons to compare their expression and subcellular distribution with endogenous DMT1 isoforms. We found endogenous DMT1 isoforms in the cytoplasm that colocalized with lysosomal-associated protein 1 (LAMP1), a marker of endolysosomes. Next, we blocked endogenous DMT1 isoforms using ebselen, a potent pharmacological inhibitor of DMT1 iron transport. Ebselen pre-treatment blocked morphine's ability to upregulate FHC protein, suggesting this pathway requires DMT1 iron transport from endolysosomes. This was further validated using viral-mediated genetic silencing of DMT1±IRE in cortical neurons, which also blocked FHC upregulation in the presence of morphine. Overall, our work demonstrates that the µ-opioid agonist morphine utilizes the endolysosomal iron transporter DMT1 to modulate neuronal cellular iron metabolism, upregulate FHC protein, and contribute to cognitive decline in neuroHIV. Morphine requires DMT1 to upregulate neuronal FHC. Cortical neurons treated with morphine release their endolysosomal iron stores to the cytoplasm and upregulate FHC, an iron storage protein associated with dendritic spine deficits and cognitive impairment in neuroHIV. This pathway requires the endolysosomal iron transporter DMT1, as pharmacological and genetic inhibitors of the transporter completely block morphine's ability to upregulate FHC. Created with BioRender.com .

Mu opioid receptor-mediated release of endolysosome iron increases levels of mitochondrial iron, reactive oxygen species, and cell death.

Objectives: Opioids including morphine and DAMGO activate mu-opioid receptors (MOR), increase intracellular reactive oxygen species (ROS) levels, and induce cell death. Ferrous iron (Fe2+) through Fenton-like chemistry increases ROS levels and endolysosomes are "master regulators of iron metabolism" and contain readily-releasable Fe2+ stores. However, mechanisms underlying opioid-induced changes in endolysosome iron homeostasis and downstream-signaling events remain unclear. Methods: We used SH-SY5Y neuroblastoma cells, flow cytometry, and confocal microscopy to measure Fe2+ and ROS levels and cell death. Results: Morphine and DAMGO de-acidified endolysosomes, decreased endolysosome Fe2+ levels, increased cytosol and mitochondria Fe2+ and ROS levels, depolarized mitochondrial membrane potential, and induced cell death; effects blocked by the nonselective MOR antagonist naloxone and the selective MOR antagonist ß-funaltrexamine (ß-FNA). Deferoxamine, an endolysosome-iron chelator, inhibited opioid agonist-induced increases in cytosolic and mitochondrial Fe2+ and ROS. Opioid-induced efflux of endolysosome Fe2+ and subsequent Fe2+ accumulation in mitochondria were blocked by the endolysosome-resident two-pore channel inhibitor NED-19 and the mitochondrial permeability transition pore inhibitor TRO. Conclusions: Opioid agonist-induced increases in cytosolic and mitochondrial Fe2+ and ROS as well as cell death appear downstream of endolysosome de-acidification and Fe2+ efflux from the endolysosome iron pool that is sufficient to affect other organelles.

Regulation of Tumor and Metastasis Initiation by Chemokine Receptors.

Tumor-initiating cells (TICs) are a rare sub-population of cells within the bulk of a tumor that are major contributors to tumor initiation, metastasis, and chemoresistance. TICs have a stem-cell-like phenotype that is dictated by the expression of master regulator transcription factors, including OCT4, NANOG, and SOX2. These transcription factors are expressed via activation of multiple signaling pathways that drive cancer initiation and progression. Importantly, these same signaling pathways can be activated by select chemokine receptors. Chemokine receptors are increasingly being revealed as major drivers of the TIC phenotype, as their signaling can lead to activation of stemness-controlling transcription factors. Additionally, the cell surface expression of chemokine receptors provides a unique therapeutic target to disrupt signaling pathways that control the expression of master regulator transcription factors and the TIC phenotype. This review summarizes the master regulator transcription factors known to dictate the TIC phenotype, along with the complex signaling pathways that can mediate their expression and the chemokine receptors that are most upstream of this phenotype.

Subsets of cancer cells expressing CX3CR1 are endowed with metastasis-initiating properties and resistance to chemotherapy.

Metastasis-initiating cells (MICs) display stem cell-like features, cause metastatic recurrences and defy chemotherapy, which leads to patients' demise. Here we show that prostate and breast cancer patients harbor contingents of tumor cells with high expression of CX3CR1, OCT4a (POU5F1), and NANOG. Impairing CX3CR1 expression or signaling hampered the formation of tumor spheroids by cell lines from which we isolated small subsets co-expressing CX3CR1 and stemness-related markers, similarly to patients' tumors. These rare CX3CR1High cells show transcriptomic profiles enriched in pathways that regulate pluripotency and endowed with metastasis-initiating behavior in murine models. Cancer cells lacking these features (CX3CR1Low) were capable of re-acquiring CX3CR1-associated features over time, implying that MICs can continuously emerge from non-stem cancer cells. CX3CR1 expression also conferred resistance to docetaxel, and prolonged treatment with docetaxel selected CX3CR1High phenotypes with de-enriched transcriptomic profiles for apoptotic pathways. These findings nominate CX3CR1 as a novel marker of stem-like tumor cells and provide conceptual ground for future development of approaches targeting CX3CR1 signaling and (re)expression as therapeutic means to prevent or contain metastasis initiation.

Interactions between chemokines: regulation of fractalkine/CX3CL1 homeostasis by SDF/CXCL12 in cortical neurons.

The soluble form of the chemokine fractalkine/CX(3)CL1 regulates microglia activation in the central nervous system (CNS), ultimately affecting neuronal survival. This study aims to determine whether CXCL12, another chemokine constitutively expressed in the CNS (known as stromal cell-derived factor 1; SDF-1), regulates cleavage of fractalkine from neurons. To this end, ELISA was used to measure protein levels of soluble fractalkine in the medium of rat neuronal cultures exposed to SDF-1. Gene arrays, quantitative RT-PCR, and Western blot were used to measure overall fractalkine expression in neurons. The data show that the rate of fractalkine shedding in healthy cultures positively correlates with in vitro differentiation and survival. In analogy to non-neuronal cells, metalloproteinases (ADAM10/17) are involved in cleavage of neuronal fractalkine as indicated by studies with pharmacologic inhibitors. Moreover, treatment of the neuronal cultures with SDF-1 stimulates expression of the inducible metalloproteinase ADAM17 and increases soluble fractalkine content in culture medium. The effect of SDF-1 is blocked by an inhibitor of both ADAM10 and -17, but only partially affected by a more specific inhibitor of ADAM10. In addition, SDF-1 also up-regulates expression of the fractalkine gene. Conversely, exposure of neurons to an excitotoxic stimulus (i.e. NMDA) inhibits alpha-secretase activity and markedly diminishes soluble fractalkine levels, leading to cell death. These results, along with previous findings on the neuroprotective role of both SDF-1 and fractalkine, suggest that this novel interaction between the two chemokines may contribute to in vivo regulation of neuronal survival by modulating microglial neurotoxic properties.

Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling.

The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.

The chemokine receptor CX3CR1 is directly involved in the arrest of breast cancer cells to the skeleton.

INTRODUCTION: Skeletal metastases from breast adenocarcinoma are responsible for most of the morbidity and mortality associated with this tumor and represent a significant and unmet need for therapy. The arrival of circulating cancer cells to the skeleton depends first on the adhesive interactions with the endothelial cells lining the bone marrow sinusoids, and then the extravasation toward chemoattractant molecules produced by the surrounding bone stroma.We have previously shown that the membrane-bound and cell-adhesive form of the chemokine fractalkine is exposed on the luminal side of human bone marrow endothelial cells and that bone stromal cells release the soluble and chemoattractant form of this chemokine. The goal of this study was to determine the role of fractalkine and its specific receptor CX3CR1 in the homing of circulating breast cancer cells to the skeleton. METHODS: We employed a powerful pre-clinical animal model of hematogenous metastasis, in which fluorescent cancer cells are identified immediately after their arrival to the bone. We engineered cells to over-express either wild-type or functional mutants of CX3CR1 as well as employed transgenic mice knockout for fractalkine. RESULTS: CX3CR1 protein is detected in human tissue microarrays of normal and malignant mammary glands. We also found that breast cancer cells expressing high levels of this receptor have a higher propensity to spread to the skeleton. Furthermore, studies with fractalkine-null transgenic mice indicate that the ablation of the adhesive and chemotactic ligand of CX3CR1 dramatically impairs the skeletal dissemination of circulating cancer cells. Finally, we conclusively confirmed the crucial role of CX3CR1 on breast cancer cells for both adhesion to bone marrow endothelium and extravasation into the bone stroma. CONCLUSIONS: We provide compelling evidence that the functional interactions between fractalkine produced by both the endothelial and stromal cells of bone marrow and the CX3CR1 receptor on breast cancer cells are determinant in the arrest and initial lodging needed for skeletal dissemination.

Role of mu-opioids as cofactors in human immunodeficiency virus type 1 disease progression and neuropathogenesis.

About one third of acquired immunodeficiency syndrome cases in the USA have been attributed to the use of injected addictive drugs, frequently involving opioids like heroin and morphine, establishing them as significant predisposing risk factors for contracting human immunodeficiency virus type 1 (HIV-1). Accumulating evidence from in vitro and in vivo experimental systems indicates that opioids act in concert with HIV-1 proteins to exacerbate dysregulation of neural and immune cell function and survival through diverse molecular mechanisms. In contrast, the impact of opioid exposure and withdrawal on the viral life cycle and HIV-1 disease progression itself is unclear, with conflicting reports emerging from the simian immunodeficiency virus and simian-human immunodeficiency virus infection models. However, these studies suggest a potential role of opioids in elevated viral production. Because human microglia, astrocytes, CD4+ T lymphocytes, and monocyte-derived macrophages express opioid receptors, it is likely that intracellular signaling events triggered by morphine facilitate enhancement of HIV-1 infection in these target cell populations. This review highlights the biochemical changes that accompany prolonged exposure to and withdrawal from morphine that synergize with HIV-1 proteins to disrupt normal cellular physiological functions especially within the central nervous system. More importantly, it collates evidence from epidemiological studies, animal models, and heterologous cell systems to propose a mechanistic link between such physiological adaptations and direct modulation of HIV-1 production. Understanding the opioid-HIV-1 interface at the molecular level is vitally important in designing better treatment strategies for HIV-1-infected patients who abuse opioids.

Opioid Modulation of Neuronal Iron and Potential Contributions to NeuroHIV.

Opioid use has substantially increased over recent years and remains a major driver of new HIV infections worldwide. Clinical studies indicate that opioids may exacerbate the symptoms of HIV-associated neurocognitive disorders (HAND), but the mechanisms underlying opioid-induced cognitive decline remain obscure. We recently reported that the µ-opioid agonist morphine increased neuronal iron levels and levels of ferritin proteins that store iron, suggesting that opioids modulate neuronal iron homeostasis. Additionally, increased iron and ferritin heavy chain protein were necessary for morphine's ability to reduce the density of thin and mushroom dendritic spines in cortical neurons, which are considered critical mediators of learning and memory, respectively. As altered iron homeostasis has been reported in HAND and related neurocognitive disorders like Alzheimer's, Parkinson's, and Huntington's disease, understanding how opioids regulate neuronal iron metabolism may help identify novel drug targets in HAND with potential relevance to these other neurocognitive disorders. Here, we review the known mechanisms of opioid-mediated regulation of neuronal iron and corresponding cellular responses and discuss the implications of these findings for patients with HAND. Furthermore, we discuss a new molecular approach that can be used to understand if opioid modulation of iron affects the expression and processing of amyloid precursor protein and the contributions of this pathway to HAND.

Morphine increases brain levels of ferritin heavy chain leading to inhibition of CXCR4-mediated survival signaling in neurons.

This study focuses on the effect of mu-opioid receptor agonists on CXCR4 signaling in neurons and the mechanisms involved in regulation of neuronal CXCR4 by opiates. The data show that CXCR4 is negatively modulated by long-term morphine treatments both in vitro and in vivo; CXCR4 inhibition is caused by direct stimulation of mu-opioid receptors in neurons, leading to alterations of ligand-induced CXCR4 phosphorylation and upregulation of protein ferritin heavy chain (FHC), a negative intracellular regulator of CXCR4. Reduced coupling of CXCR4 to G-proteins was found in the brain of morphine-treated rats, primarily cortex and hippocampus. CXCR4-induced G alpha(i)/G betagamma activities were suppressed after 24 h treatment of cortical neurons with morphine or the selective mu-opioid agonist DAMGO (D-Ala2-N-Me-Phe(4)-glycol(5)-enkephalin), as shown by analysis of downstream targets of CXCR4 (i.e., cAMP, Akt, and ERK1/2). These agonists also prevented CXCL12-induced phosphorylation of CXCR4, indicating a deficit of CXCR4 activation in these conditions. Indeed, morphine (or DAMGO) inhibited prosurvival signaling in neurons. These effects are not attributable to a reduction in CXCR4 expression or surface levels but rather to upregulation of FHC by opioids. The crucial role of FHC in inhibition of neuronal CXCR4 was confirmed by in vitro and in vivo RNA interference studies. Overall, these findings suggest that opiates interfere with normal CXCR4 function in the brain. By this mechanism, opiates could reduce the neuroprotective functions of CXCR4 and exacerbate neuropathology in opiate abusers who are affected by neuroinflammatory/infectious disorders, including neuroAIDS.

Alterations of CXCR4 function in µ-opioid receptor-deficient glia.

The chemokine receptor CXCR4 and the µ-opioid receptor (MOR) are G-protein-coupled receptors that are essential for normal function of the nervous and immune systems. Several studies have suggested that MOR is a key regulator of CXCR4 in the brain; however, the molecular basis of the opioid-chemokine interaction is not fully understood, and it may involve different mechanisms in neuronal and glial cells. Our previous studies demonstrated that MOR stimulation specifically upregulates the protein ferritin heavy chain - an inhibitor of CXCR4 - in neurons, and suggested that additional mechanisms could be operative in glia. In this study, we investigated CXCR4 function in brains and astroglial cultures deprived of MOR. Reduced coupling of CXCR4 to G-proteins was found in brain slices and tissue homogenates of MOR(-/-) mice as compared with wild-type controls. CXCR4-induced signaling was also reduced in glial cultures from MOR(-/-) mice, as shown by analysis of CXCR4 downstream targets (Akt and ERK1/2). Pharmacological studies with δ-opioid receptor (DOR)-specific ligands suggested that DOR-CXCR4 interactions are implicated in the inhibition of CXCR4 in MOR-deficient cells both in vitro and in vivo. Moreover, increased CXCR4/DOR co-immunoprecipitation was found in brain tissue and cultured glia from MOR(-/-) mice. Importantly, CXCR4 function was restored by pretreatment with a DOR antagonist. Overall, these findings indicate that DOR plays a crucial role in the regulation of CXCR4 in glia, probably via silent receptor heterodimers. The data also suggest that the opiate system interferes with normal CXCR4 function in different ways, depending on receptor subtypes.

CXCL12-induced rescue of cortical dendritic spines and cognitive flexibility.

Synaptodendritic pruning is a common cause of cognitive decline in neurological disorders, including HIV-associated neurocognitive disorders (HAND). HAND persists in treated patients as a result of chronic inflammation and low-level expression of viral proteins, though the mechanisms involved in synaptic damage are unclear. Here, we report that the chemokine CXCL12 recoups both cognitive performance and synaptodendritic health in a rodent model of HAND, which recapitulates the neuroinflammatory state of virally controlled individuals and the associated structural/functional deficiencies. CXCL12 preferentially regulates plastic thin spines on layer II/III pyramidal neurons of the medial prefrontal cortex via CXCR4-dependent stimulation of the Rac1/PAK actin polymerization pathway, leading to increased spine density and improved flexible behavior. Our studies unveil a critical role of CXCL12/CXCR4 signaling in spine dynamics and cognitive flexibility, suggesting that HAND - or other diseases driven by spine loss - may be reversible and upturned by targeting Rac1-dependent processes in cortical neurons.

COVID-19 and possible links with Parkinson's disease and parkinsonism: from bench to bedside.

This Viewpoint discusses insights from basic science and clinical perspectives on coronavirus disease 2019 (COVID-19)/severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection in the brain, with a particular focus on Parkinson's disease. Major points include that neuropathology studies have not answered the central issue of whether the virus enters central nervous system neurons, astrocytes or microglia, and the brain vascular cell types that express virus have not yet been identified. Currently, there is no clear evidence for human neuronal or astrocyte expression of angiotensin-converting enzyme 2 (ACE2), the major receptor for viral entry, but ACE2 expression may be activated by inflammation, and a comparison of healthy and infected brains is important. In contrast to the 1918 influenza pandemic and avian flu, reports of encephalopathy in COVID-19 have been slow to emerge, and there are so far no documented reports of parkinsonism apart from a single case report. We recommend consensus guidelines for the clinical treatment of Parkinson's patients with COVID-19. While a role for the virus in causing or exacerbating Parkinson's disease appears unlikely at this time, aggravation of specific motor and non-motor symptoms has been reported, and it will be important to monitor subjects after recovery, particularly for those with persisting hyposmia.